Avian reoviruses are members of the genus Orthoreovirus, one of the 12 genera of the family Reoviridae (Attoui et al., 2000, J. Gene. Virol. 81:1507-15). These viruses are important bird pathogens and cause significant economical losses in poultry farming industry (Jones, 2000. Rev. Sci. Tech. 19: 614-25). Avian reoviruses are viruses without lipid envelope which replicate in the cytoplasm of the infected cells and have a genome with 10 segments of double stranded RNA surrounded by two concentric protein shells of 85 nm in diameter (Zhang et al., 2005. Virology, 343: 25-35). Their genomic segments are divided into three classes according to their electrophoretic mobility, three of class L (large), another three of class M (medium) and four of class S (small) (Varela and Benavente, 1994. J. Virol. 68: 6775-7). With the exception of the tricistronic segment S1, all the other genes are monocistronic (Bodelon et al., 2001. Virology, 290: 181-91). The genomic segments are translated by means of an RNA-dependent polymerase to produce messenger RNAs (mRNA) with a nucleotide sequence identical to that of the positive strand of the segment of double stranded RNA (Li et al., 1980. Virology, 105: 41-51). Viral mRNAs perform two functions in the infected cells: they program the viral protein synthesis in the ribosomes and serve as a template for synthesizing negative strands of the genomic segments.
The genome of avian reovirus encodes at least 12 proteins, 8 of which are structural proteins (which are incorporated into virion), and 4 non-structural proteins which are expressed in infected cells but do not form part of the mature reovirions (Martínez-Costas et al., 1997. J. Virol. 71: 59-64). The proteins encoded by class L genes are called lambda (λ), those encoded by class M genes are called mu (μ) and those encoded by class S genes are called sigma (σ). An alphabetical suffix (λA, λB, etc.) has been assigned to the structural proteins of each class according to their electrophoretic mobility. Reovirion contains at least 10 different structural proteins different, 8 of which (λA, λB, λC, μA, μB, σA, σB and σC) are primary products from their mRNA translation, whereas the other two, μBN and μBC resulted from the proteolytic processing of the precursor μB (Varela et al., 1996. J. Virol. 70: 2974-81). In addition to the structural proteins, avian reoviruses express four non-structural proteins. Therefore, genes M3 and S4 express two major non-structural proteins called μNS and σNS, respectively (Varela and Benavente, 1994, mentioned ad supra) whereas p10 and p17 are encoded by the first two cistrons of the S1 gene (Bodelon et al., 2001, mentioned ad supra).
Avian reoviruses replicate in globular cytoplasmic inclusions called viral factories or viroplasmas which contain structural and non-structural viral proteins, however they lack membranes and cellular organelles (Touris-Otero et al., 2004; J. Mol. Biol. 341: 361-74). The individual expression of viral proteins in transfected cells revealed that non-structural muNS protein is the only protein of the avian reovirus capable of forming inclusions when it is expressed in the absence of other viral factors (Touris-Otero et al., 2004; mentioned ad supra). This, and the fact that the globular cytoplasmic inclusions formed by muNS in transfected cells are very similar in appearance to the viral factories of infected cells, suggest that muNS is the minimum viral factor required for forming viral factories in infected cells with avian reovirus. The analysis of transfected cells that co-express muNS and other viral proteins revealed that muNS plays an important role in the early steps of virus morphogenesis and that the recruitment of avian reovirus proteins into the viral factories is a selective and temporally controlled process (Touris-Otero et al., 2004; mentioned ad supra).
Mammalian reovirus also replicate in globular cytoplasmic inclusions. Like the avian reoviruses, the non-structural muNS protein has been found to be involved in inclusion formation, as well as in the recruitment of other components into the inclusions for possible involvements in genome replication and in particle assembly.
Despite the fact that avian and mammalian reovirus muNS proteins show only 28.3% of sequence identity, they both contain two regions in their C-terminus end with a high “coiled-coil” structure probability. On the other hand, the mammalian protein is 86 amino acids longer and is capable of making more primary contacts with other structural and non-structural viral proteins than the avian protein (Broering et al. 2004; J. Virol. 78: 1882-92). Even though the muNS proteins of all mammal reovirus (MRV) strains produce globular inclusions when they are expressed in transfected cells, most of the strains produce viral factories with filamentous morphology during infection (Parker et al. 2002; J. Virol. 76:4483-96; Broering et al., 2002 J. Virol. 76: 8285-8297). The filamentous phenotype of the mammalian reovirus factories has been attributed to the mu2 protein, due to its capacity to associate both with microtubules and with mammalian reovirus muNS. The expression of the truncated versions of MRV muNS in transfected cells revealed that the segment between the residues 471-721 is the smallest region of muNS necessary and sufficient for forming inclusions (Broering et al. 2005; J. Virol. 79: 6194-6206). It is predicted that this region contains two segments of sequences with high “coiled-coil” structure-forming probability, which are bound by a region, preceded by a section of approximately 50 residues and followed by a C-terminus tail. Despite the fact that minimum region of muNS in MRV capable of forming inclusions has been described, said region has not been identified in avian reoviruses. In the present specification, in addition to determining said region, muNS domains capable of being incorporated into the cytoplasmic inclusions formed by the whole protein is described to check which of the domains are directly involved in the interaction between the monomers of muNS and to thus develop a method for purifying proteins, as well as a method for detecting the interaction between polypeptides.
There are several systems designed today for determining protein interaction of which the double hybrid system is the most popular. This system is based on the expression of two fusion proteins: one in which the X protein is fused to the DNA-binding domain of the transcription factor GCN4; and another in which the Y protein is fused to the transcription activation domain of the same factor GCN4. If X and Y interact, they are expected to reconstruct a functional GCN4 in the cell which will activate the transcription of a reporter gene. The most obvious problems of this system include: i) even though X and Y interact, the architecture of said interaction does not usually allow reconstructing a functional GCN4; ii) the fusions may alter the structures of the different GCN4 domains or of the interaction domains between the test proteins.
A new system using the formation of inclusions by mammalian reovirus muNS protein as a platform for detecting interactions between proteins in vivo in mammalian cells has been described recently (Miller et al., 2007. Mol Cell Proteomics. 6, 1027-38) and it has also been adapted for use in yeasts (Schmitz et al., 2009. Nat Methods; 6, 500-2). In this system, the test protein fuses with the C-terminus area of muNS so that the fusion generates cytoplasmic inclusions and attracts ligand of the test protein thereto. In the yeast system, these authors show that their system is better than the double hybrid system in the number and type of interactions detected, at least with the proteins assayed in said research. However, this system has several problems which include: i) certain proteins may fold incorrectly when fused with muNS-Mi and loss capacity to interact with their ligands; ii) some proteins may interfere with muNS-Mi inclusion-forming capacity and, do not form inclusions or generate intracellular aggregates, the detection of interactions of being largely altered; iii) the intracellular location of the test protein or the ligand may not be suitable to enable detecting same in cytoplasmic inclusions.
Therefore, there is a need in the state of the art to develop a system having advantages with respect to the existing systems, in which for example, the protein fused to the inclusions does not alter the formation of said inclusions, the fused protein maintains its activity and several epitopes can be included in said inclusions.